Bidet toilet seats with warm-water tanks: residual chlorine, microbial community, and structural analyses

Despite the reported health-related advantages of the use of warm water in bidets, there are healthrelated disadvantages associated with the use of these toilet seats, and the bacterial research is sparse. We conducted a survey on the hygienic conditions of 127 warm-water bidet toilet seats in restrooms on a university campus. The spray water from the toilet seats had less residual chlorine than their tap water sources. However, the total viable microbial count was below the water-quality standard for tap water. In addition, the heat of the toilet seats’ warm-water tanks caused heterotrophic bacteria in the source tap water to proliferate inside the nozzle pipes and the warmwater tanks. Escherichia coli was detected on the spray nozzles of about 5% of the toilet seats, indicating that the self-cleaning mechanism of the spray nozzles was largely functioning properly. However, Pseudomonas aeruginosa was detected on about 2% of the toilet seats. P. aeruginosa was found to remain for long durations in biofilms that formed inside warm-water tanks. Infectionprevention measures aimed at P. aeruginosa should receive full consideration when managing warmwater bidet toilet seats in hospitals in order to prevent opportunistic infections in intensive care units, hematology wards, and other hospital locations. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/wh.2015.057 om https://iwaponline.com/jwh/article-pdf/14/1/68/394577/jwh0140068.pdf 2020 Toru Iyo (corresponding author) Department of Health Sciences, School of Allied Health Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan E-mail: iyot.ahs@kitasato-u.ac.jp Keiko Asakura Department of Social and Preventive Epidemiology, School of Public Health, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan Makiko Nakano Mutsuko Yamada Kazuyuki Omae Departments of Preventive Medicine and Public Health, Kieo University School of Medicine, 35 Shinanomachi, Sinjuku-ku, Tokyo 160-8582, Japan


INTRODUCTION
Warm-water bidet toilet seats are equipped with a device that sprays warm water (spray water) on the external genitalia and anus after urination and defecation. A toilet seat equipped with bidet functions first appeared on the market in the United States in 1964 (Altman ). This was followed by the development of functional electric warm-water bidet toilet seats in Japan, of which at least 30 million units have been sold nationwide to date (Hasegawa ). A consumer behavior survey conducted by the Japanese Cabinet Office showed that 74.0% of households had warm-water bidet toilet seats in March 2013, with 102.9 units per 100 households (effectively one per household) (Cabinet Office Government of Japan ). Broadly, there are two types of warm-water bidet toilet seats: tank types and on-demand types. In the tank type, the spray water is warmed to a suitable temperature in a tank, whereas in the on-demand type the water is warmed as needed inside a tube. Tank-type products are both cheaper and more common than on-demand types in Japan. In the United States, consumers have generally resisted the use of warm-water bidet toilet seats, with only around 200,000 units sold in the past 45 years (Altman ).
The use of warm-water bidet toilet seats has been reported to promote defecation in people with spinal injuries (Uchikawa et  While hygienic evaluations of the warm water sprayed from bidet toilet seat nozzles are important to consider from the perspective of public health, there has been almost no bacterial research conducted on these toilet seats to date. The water sprayed from bidet toilet seats is disinfected by the residual chlorine in tap water, which is the source of the bidet spray water. However, we hypothesized that the heat of the warm-water tank could eliminate the residual chlorine, thus negating the effect of this important built-in factor for disinfection. Therefore, we surveyed the state of residual chlorine and microbial indicators in the spray water of warm-water tanks of bidet toilet seats at the campus of Kitasato University, Kanagwa, Japan. We also evaluated the disinfection status and microbial hygiene of the spray water.

Warm-water bidet toilet seats
To evaluate the hygiene status of bidet toilet seats with warm-water tanks (hereafter, warm-water bidet toilet seats), a survey of their microbial communities was conducted at the Sagamihara campus of Kitasato University in 2012 and 2013. Of the total 127 seats analyzed, there were 43 toilet seats for men's use, 71 for women's use, and 13 for barrier-free use. The campus contains both university and hospital buildings. Of the toilet seats studied, 86 were in a research building (33 men's, 45 women's, eight barrier-free) and 41 were in an outpatient hospital building (10 men's, 26 women's, 10 barrier-free). Between about 2 to 10 years had passed since these toilet seats had been installed, and all were functioning properly.

Spray water
Residual chlorine and microorganism indicators in the spray water from the warm-water bidet toilet seats were surveyed twice. For the first survey, spray water was collected directly as it came out of the nozzle, and tap water was used as a control. In the second survey, the surface of the nozzle was first disinfected with 70% ethanol and then rinsed with sterile purified water so that the sample could be collected without any contamination from the nozzle surface. About 200 mL of spray water from the nozzle was collected in sterilized bottles (containing 30 mg sodium thiosulfate) for microorganism testing.
About 50 mL of spray water was collected in sterilized bottles for residual chlorine testing. The samples were kept at 4 W C in refrigerated storage until analysis. Measurements were performed quickly.

Tap water
Tap water for control specimens was collected from faucets in the restrooms that were surveyed. To eliminate as much contamination as possible on the faucet, the tap water was allowed to run for about 1 min before samples (∼200 mL) were collected for microorganism testing in sterilized bottles (containing 30 mg sodium thiosulfate). Next, about 50 mL of tap water was collected for residual chlorine testing in sterilized bottles. The samples were kept at 4 W C in refrigerated storage until analysis. Measurements were performed quickly.

Residual chlorine
For spray water and tap water samples, residual chlorine levels (mg/L) were measured using a DR2010 multi-item rapid water-quality measuring instrument (Hach Co.; Tilamook, OR, USA) with the N,N-Diethyl-p-phenylenediamine (DPD) method (USEPA-approved Hach Method 8167).   Tokyo, Japan) and sequence analysis was performed with an Applied Biosystems 3730xl sequencer (Applied Biosystems;

Total viable count
Foster City, CA, USA).

Statistical analysis
The geometric means (GM) and geometric standard deviations were calculated for total viable count, HPC, and residual chlorine. When there were non-detected data, the detection limit was assigned for use in calculation. Statistical comparisons of total viable count, HPC, and residual chlorine in spray water and tap water were performed using

Residual chlorine
The GM of residual chlorine in the spray water of warm-water bidet toilet seats and in tap water was 0.04 mg/L and 0.24 mg/ L, respectively, which represented a significant difference (P < 0.01). This result demonstrated that there is clearly less residual chlorine in spray water than in tap water (Table 1).
Moreover, when these data were log-normalized, it became clear that artificial factors (e.g., heating) caused the residual chlorine to disappear in the spray water. However, no significant difference in the residual chlorine concentration of spray water was observed between the male and female restrooms.
Residual chlorine levels were higher in the spray water from toilet seats in the outpatient building than from those in the research building (P < 0.01; Figure 1). Frequent inflow of tap water into a toilet's warm-water tank is needed to maintain the chlorine concentration in spray water; that is, the residual chlorine concentration cannot be maintained without frequent use. As such, we surmised that the toilet seats in the outpatient building were used more often than those in the research building.

Total viable count and HPC in spray water
The total viable count and HPC of spray water from warmwater bidet toilet seats and control tap water are shown in Table 2. The total viable count and HPC were both significantly higher in spray water than in tap water, and the difference was particularly apparent for HPC, with an increase of around 2-3 log10 (P < 0.01). Detection rates were also higher in spray water than in tap water (P < 0.01). Significant differences in total viable count were not observed between male and female restrooms (Table 2) or between the research and outpatient buildings (data not shown).
A significant difference in HPC was observed between the male and female restrooms (P < 0.01) ( Figure 2). Examining the data by building, a significant difference was observed between the male and female restrooms in the research building, but not in the outpatient building (data not shown). In addition, HPC was clearly higher in the outpatient building's male restrooms than in the male restrooms in the research building (P < 0.01; Figure 3).

Relationship of residual chlorine with total viable count and HPC
The correlations of residual chlorine with total viable count and HPC are shown in Figures 4 and 5, respectively. The threshold of detection for total viable count ( 100 CFU/mL) was around a residual chlorine level of 0.1 mg/mL, while the threshold of detection for HPC ( 1,000 CFU/mL) was around a residual chlorine level of 0.2 mg/mL.
Residual chlorine levels were significantly negatively correlated with both total viable count and HPC. The Spearman's rank correlation coefficient for residual chlorine and  Effect of nozzle cleaning on the survey results     was collected directly or following disinfection of the nozzle, respectively. These results indicate that the total viable count and HPC decreased when spray water was collected after cleaning the nozzle (P < 0.01).

Detection of fecal indicator bacteria and P. aeruginosa
Surveys of fecal indicator bacteria and P. aeruginosa in the spray water were conducted from early September to early October (for the first survey) and from late October to late November (for the second survey). The detection rates of fecal indicator bacteria and P. aeruginosa were nearly identical in both surveys (Table 3). Coliform bacteria were detected in the spray water collected from seven toilet seats (5.5%), E. coli was detected in the water from three toilet seats (2.4%), enterococci were detected in the water from four toilet seats (3.1%), and either E. coli or enterococci were detected in the water from six toilet seats (4.7%). When tap water was analyzed as a negative control, no fecal indicator bacteria were detected, as expected.
A Chi-squared test of the fecal indicator bacteria detection rates in male and female restrooms showed a tendency for coliform bacteria to be detected more often in spray water from toilet seats in male restrooms (P < 0.05) than in female restrooms. Most fecal indicator bacteria were detected at a concentration of 1 MPN/mL or less ( In the first and second surveys, P. aeruginosa was specifically detected in water from the same male and female toilet seats. Of the 127 warm-water bidet toilet seats, P. aeruginosa was detected in the spray water of only two seats ( Table 4) at concentrations of 5 CFU/mL (male restroom) and 10 CFU/mL (female restroom).

Infectious microorganisms
Neither Salmonella, V. parahaemolyticus, B. cereus, nor S. aureus was detected in the spray water from the warmwater bidet toilet seats or in tap water.
Microbial community structure Figure 7 shows the microbial community structure in spray water collected from five warm-water bidet toilet seats on the university campus (spray waters 1-5, lanes 1-5) and from one toilet in a residence in the same city, which was evaluated as a control (spray water 6, lane 6).   (Table 5).
The bacterial groups that the spray water samples had in common were Arthrobacter, a type of actinomycete; Novosphingobium, a soil bacterium; Sphingomonas; and Sphingopyxi. Phylogenetic analysis with the neighbor-joining method revealed that Novosphingobium (bands b2, b5) and Sphingomonas (bands c1, c3-c6) have similar DNA sequences (Figure 8).

PCR-DGGE isolation and sequence analysis and
identification indicated that the environmental microorganisms found in trace amounts in tap water create biofilms and proliferate inside the warm-water tanks of  bidet toilet seats, and are then discharged into the spray water.

DISCUSSION
The ability to maintain and regulate residual chlorine is an important built-in factor for maintaining the hygiene and disinfection of spray water. In this study, we surveyed 127 warm-water bidet toilet seats and found that the concentration of chlorine decreased due to heating and remaining stagnant for long periods. Since there was no obvious change in the toilet seats surveyed over time, the observed decrease in residual chlorine was not due to deterioration of the toilet seats themselves.
The effectiveness of disinfection depends on the CT value, which is the product of residual chlorine concentration and contact time (Zamyadi et al. , ). When water is sprayed from a bidet toilet seat's nozzle, the tap water flows in to replace it, thereby increasing the amount of residual chlorine in the warm-water tank. However, if a toilet seat is not used from nighttime to the next morning, the water in the tank will stay warm without any new influx of tap water, causing the residual chlorine levels in the tank to decrease. Therefore, a warm-water bidet toilet seat's frequency of use and the interval between uses will affect the amount of residual chlorine in the warm-water tank as well as the contact time, thereby changing the CT value.
When heterotrophic bacteria are cultured at low temp-   spray water showed a marked increasing trend, rising to levels 100 to 1,000 times higher than those found in tap water. This suggests that microorganisms repopulate and biofilms form at higher rates in the warm-water tanks and internal tubing of bidet toilet seats than in tap water pipes due to heating, and because of remaining stagnant for long periods.
Furthermore, the nozzles of warm-water bidet toilet seats can become contaminated with feces because they are used to wash the area around the anus after defecation. including its source, behavior inside warm-water tanks, ability to survive for long periods, and multidrug resistance.
As total viable count is a widely used indicator of hygiene and disinfection, our results indicate that the disinfection effect was clearly lower in spray water than in tap water (Codony et al. ). Although residual chlorine decreased and total viable count and HPC increased in the spray water, there were no marked increases in the amount or detection frequency of fecal indicator bacteria and P. aeruginosa. Therefore, hygienic safety was being maintained overall, as the concentrations of total viable bacteria, fecal indicator bacteria, and P. aeruginosa were low.
PCR-DGGE isolation and sequence analysis and identi-

DISCLOSURE
The authors declare no conflicts of interest. Supported by the Japan Toilet Seat Appliances Association.